1. Technical Field
The present invention relates generally to biological assays, and more particularly to a method and system for identifying and monitoring nitrosothiol moieties in fluidized biological samples.
2. Description of Related Art
Nitric oxide (NO) is a molecule, which is biologically important in both a physiological and a pathological sense and implicated in biological processes, including control of systemic blood pressure, respiration, digestion, platelet aggregation and cerebral blood flow, as well as contributing to the microbicidal and tumouricidal activities of macrophages. Nitric oxide is an endogenous bioactive molecule produced by the conversion of L-arginine to L-citrulline by the nitric oxide synthase (NOS) family of enzymes.
Under physiological conditions, NO is unstable and easily reacts with oxygen to form oxides of nitrogen (NOx). Thus, it is difficult to measure levels of nitric oxide because it exists as a free radical with a short half-life of approximately 10 to 30 seconds in aqueous solution and approximately 0.46 ms in whole blood (Feelisch, M, Stampler, J. S., Methods in Nitric Oxide Research, John Wiley & Sons, New York, 1st Edition 49-65 (1996)). Consequently, many of the reports implicating NO in various physiological processes and many disease states are based on the measurement of NO metabolites or NO containing compounds. These NO containing compounds include S-nitrosothiols that are known to be formed in vivo by S-nitrosation of thiol-containing proteins and peptides, such as albumin and hemoglobin, as well as smaller molecular weight compounds such as cysteine, glutathione, homocysteine, etc.
S-nitrosothiols are endogenous biologically active nitric oxide containing compounds in which nitric oxide in the form of nitrosonium ion, NO+, is attached to the free sulfhydryl group of a cysteine residue. S-nitrosothiols exist as both small (S-nitrosocysteine, S-nitrosoglutathione, etc.) and large (such as S-nitrosoalbumin, nitrosylated proteins, etc.) molecular weight compounds and can be found at various levels throughout the body where they are capable of initiating signaling processes similar to as well as distinct from those associated with the NO radical.
Recent studies suggest that alterations in biological S-nitrosothiol levels are linked to asthma, inflammation, hypertension, apoptosis and atherosclerosis and are predictive of adverse outcomes. For example, Corradi et al. (American Journal of Respiratory and Critical Care Medicine, “Increased Nitrosothiols in Exhaled Breath Condensate in Inflammatory Airway Disease” V. 163: pp. 854-858 (2001) found that S-nitrosothiol levels increased in patients with severe asthma; Tyurin et al., (Circulation Research, “Elevated Levels of S-Nitrosoalbumin in Preeclampsia Plasma” V. 88: p. 1210, (2001)) determined that plasma concentration of S-nitrosothiols was much higher in women with preeclampsia then women experiencing a normal pregnancy; and Massey, et al. (J. Am. Soc. Nephrol., “Increase Plasma S-Nitrosothiol Concentration Predict Cardiovascular Outcomes among Patients with End-Stage Renal Disease” V. 15: pp. 470-476 (2004)) determined that levels of S-nitrosothiols, are increased among patient undergoing chronic hemodialysis and this high level seems to be due to reduced breakdown of the S-nitrosothiols which causes less bioavailability of the molecule to the patient and contributes to increased negative cardiovascular events.
Therefore, with the increased levels of nitrosothiols shown to correlate with multiple disease states, the ability to functionally assess these levels in biological fluids represents a valuable tool for aiding in the diagnosis and treatment of disease. At present, there exist several methods for assaying nitrosothiol levels in biological fluid samples. The most popular is the Saville assay that is based on mercury ion-mediated heterolytic cleavage of the S—NO bond. Other methods include uv/visible spectroscopy, fluorescence spectroscopy, and the high performance liquid chromatography (HPLC) analysis, which can be combined with chemiluminescence or an electrochemical detector. However, these assays are limited in their current clinical application by overly complex methodologies, cost-ineffective material requirements, insufficient sensitivity and low throughput and/or assay duration.
Therefore, there exists a need for a clinically applicable assay to determine the level of S-nitrosothiols that uses a simple methodology that is convenient, scalable, cost effective, and reproducible with sufficient specificity and sensitivity to accurately measure the level of nitrosothiol in fluidized samples without unwanted artifacts.